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Comprehensive study on Escherichia coli genomic expression : does position really matter?

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Abstract
As a biorefinery platform host, Escherichia coli has been used extensively for the production of metabolites with commercial interest. Moreover, promoter and terminator databases are readily available as well as a wide amount of expression vectors and numerous gene editing technologies. Together with the ever-reducing cost of synthetic DNA, the range of possibilities in metabolic engineering is expanding even more. Recent advances have secured the possibility of integrating whole synthetic pathways with ease and high efficiency onto the bacterial genome, hereby overcoming the need for plasmid expression and their associated instability. However, the question of where and how to incorporate your novel optimized pathway remains unanswered. To address this issue, we first improved the integration methodology of genes and pathways into the chromosome. To this end we created a serine integrase recombinational engineering (SIRE) system that allows fast and highly efficient integration in the genome, in single and in multiple copies. We were able to introduce a 10.3 kb biosynthetic gene cluster on different locations throughout the genome with an efficiency of 100% for the integrating step and without the need for selection markers on the knock-in cassette (PMID: 30345503). This methodology allowed us to comprehensively investigate multiple factors that can potentially influence gene expression in an organisms’ chromosome. The tested factors are: 1) chromosomal locations, spread over the E. coli macrodomains, 2) the influence of carbon and nitrogen source on gene expression, 3) the effect of supercoiling on gene expression, 4) the effect of gene direction in relation to the DNA replication fork and 5) the effect of induced burden on chromosomal expression. To enable these experiments, we have designed fluorescent expression cassettes that eliminate specific local effects on gene expression originating from the surrounding genes, transcriptional read through and influence from transcription factors. Overall, we can conclude from this study that characterising multiple effects on genomic expression is crucial in the development of production hosts, in order to maximize the range in expression of pathway genes. The availability of libraries of strains with different expression levels can only augment the possibilities for the development of new production hosts.

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Chicago
Goormans, Anke, Nico Snoeck, Hannes Decadt, Gert Peters, Pieter Coussement, Dries Van Herpe, Joeri Beauprez, Sofie De Maeseneire, and Wim Soetaert. 2019. “Comprehensive Study on Escherichia Coli Genomic Expression : Does Position Really Matter?” In Renewable Resources and Biorefineries 15, Abstracts.
APA
Goormans, A., Snoeck, N., Decadt, H., Peters, G., Coussement, P., Van Herpe, D., Beauprez, J., et al. (2019). Comprehensive study on Escherichia coli genomic expression : does position really matter? Renewable Resources and Biorefineries 15, Abstracts. Presented at the 15th International conference on Renewable Resources and Biorefineries (RRB-15).
Vancouver
1.
Goormans A, Snoeck N, Decadt H, Peters G, Coussement P, Van Herpe D, et al. Comprehensive study on Escherichia coli genomic expression : does position really matter? Renewable Resources and Biorefineries 15, Abstracts. 2019.
MLA
Goormans, Anke et al. “Comprehensive Study on Escherichia Coli Genomic Expression : Does Position Really Matter?” Renewable Resources and Biorefineries 15, Abstracts. 2019. Print.
@inproceedings{8618431,
  abstract     = {As a biorefinery platform host, Escherichia coli has been used extensively for the production of metabolites with commercial interest. Moreover, promoter and terminator databases are readily available as well as a wide amount of expression vectors and numerous gene editing technologies. Together with the ever-reducing cost of synthetic DNA, the range of possibilities in metabolic engineering is expanding even more. Recent advances have secured the possibility of integrating whole synthetic pathways with ease and high efficiency onto the bacterial genome, hereby overcoming the need for plasmid expression and their associated instability. However, the question of where and how to incorporate your novel optimized pathway remains unanswered. 
To address this issue, we first improved the integration methodology of genes and pathways into the chromosome. To this end we created a serine integrase recombinational engineering (SIRE) system that allows fast and highly efficient integration in the genome, in single and in multiple copies. We were able to introduce a 10.3 kb biosynthetic gene cluster on different locations throughout the genome with an efficiency of 100% for the integrating step and without the need for selection markers on the knock-in cassette (PMID: 30345503).
This methodology allowed us to comprehensively investigate multiple factors that can potentially influence gene expression in an organisms’ chromosome. The tested factors are: 1) chromosomal locations, spread over the E. coli macrodomains, 2) the influence of carbon and nitrogen source on gene expression, 3) the effect of supercoiling on gene expression, 4) the effect of gene direction in relation to the DNA replication fork and 5) the effect of induced burden on chromosomal expression. To enable these experiments, we have designed fluorescent expression cassettes that eliminate specific local effects on gene expression originating from the surrounding genes, transcriptional read through and influence from transcription factors.
Overall, we can conclude from this study that characterising multiple effects on genomic expression is crucial in the development of production hosts, in order to maximize the range in expression of pathway genes. The availability of libraries of strains with different expression levels can only augment the possibilities for the development of new production hosts.},
  author       = {Goormans, Anke and Snoeck, Nico and Decadt, Hannes and Peters, Gert and Coussement, Pieter and Van Herpe, Dries and Beauprez, Joeri and De Maeseneire, Sofie and Soetaert, Wim},
  booktitle    = {Renewable Resources and Biorefineries 15, Abstracts},
  language     = {eng},
  location     = {Toulouse, France},
  title        = {Comprehensive study on Escherichia coli genomic expression : does position really matter?},
  year         = {2019},
}